Recent observations have suggested that the fine details of protein synthesis may be somewhat different than the standard 80S initiation pathway. The first observation is that factor concentrations can influence start site selection in a variety of different mRNAs. Although this has been reported previously or elF2, elF4F and elF5, we report this also for elF5B. The second is that internal initiation appears to be less sensitive to regulation via phosphorylation of elF2 (at least for the EMCV IRES). Reduction in 40S.ternary complex levels by heme deficiency, dsRNA or treatment with the interferon inducible protein p56 fails to inhibit IRES-mediated expression of a reporter protein from the bicistronic mRNA pGEM-CAT-EMCV-LUC while cap-dependent translation is quite sensitive. The third is the observation that yeast which lack elF2A show an up-regulation of expression from the Ure2p IRES. Based upon information in the database, elF2A mRNA is down-regulated under all conditions of stress. With the general observation that many stress response proteins are up-regulated at times when elF4F activity is down-regulated, these proteins are thought (or have been shown) to be expressed via internal initiation. Thus, the regulated expression via cellular IRES elements may reflect levels of both elF4F and elF2A activity. These three basic observations have led to the following specific aims: 1. extend the biochemical analysis of the 80S pathway 2. determine the mechanism of IRES-mediated translation initiation 3. determine the influence of translation factor concentrations on start site selection 4. determine the role of elF2A in internal initiation As an extra characteristic of start site selection, we would like to determine the kinetics of ribosome scanning with the desired outcome being that we can then define the "motor" for scanning and that by altering the rate of scanning, perhaps influence start site selection. Secondly, it should be possible to define the factor requirements for those factors not already present in the initial 48S complex prior to scanning or for which additional copies might be required (i.e. elF4F, elF4A and elF4B are good candidates here).